The past decade has seen a veritable explosion in our knowledge of cardiac membrane conductances, largely due to the ability to obtain healthy isolated cardiac myocytes. Most of this new information has been obtained from ventricular and atrial myocytes because of the ease with which they are dissociated. In contrast much less progress has been made on the Purkinje myocyte although examining its properties is central to understanding the origin of many life threatening arrhythmias. Our laboratory has developed a reliable technique for the dissociation of Purkinje myocytes from their collagenous matrix. We study the electrophysiologic properties of the isolated Purkinje myocytes and also those of the Purkinje fibers. In the present application we propose to continue our studies of three Purkinje membrane currents: the inward rectifier current iK1, the pacemaker to a number of important questions. With respect to iK1 these questions include the origin or rectification, the role played by internal K in gating iK1, and the control of the inactivation of this conductance by B stimulation. Our investigations of the pacemaker current will center on our recent finding that acetylcholine can reverse B agonist effects on if without having direct effects of its own. We will also investigate the origin of the pacemaker activation delay. Our studies of the Na/K pump current will attempt to elucidate the reasons for an order of magnitude difference in the ability of dihydroouabain to inhibit the Na/K pump current in ventricular versus Purkinje myocytes. A second set of experiments examines the interaction of the Na/K pump with the Na/Ca exchanger in response to a calcium load, or in response to prolonged blockade of the Na/K pump by cardiac glycosides. These studies should provide us with important insights into the normal electrophysiology and pharmacology of the Purkinje myocyte, and how this normal function can be modified.